For diagnostic applications in particular, the target nucleic acid sequence may be only a minute portion of the total pool of DNA or RNA in a sample to be screened, so that it may be difficult to detect the presence of the target nucleic acid sequence using nonisotopically labeled or end-labeled oligonucleotide probes. Thus, diagnostic tests employing DNA probes to detect rare species of nucleic acids are often not sensitive enough to be practical for use outside of the research laboratory.
One attempt to overcome the sensitivity problem is the polymerase chain reaction (PCR) method, described in U.S. Pat. Nos. 4,683,195 and 4,683,202 ("the '195 and '202 patents"). This method proceeds basically, as follows:
a) treating a sample suspected of containing the target nucleic acid sequence of interest with one oligonucleotide primer for each strand of the target nucleic acid sequence, under hybridizing conditions and in the presence of a polymerase, e.g., the Klenow fragments of Escherichia coli DNA polymerase I, such that an extension product of each primer is synthesized if the target nucleic acid sequence is present; PA1 b) placing the sample after step (a) under denaturing conditions to separate any primer extension products that are synthesized from the templates on which they are synthesized to produce single-stranded molecules; PA1 c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under the conditions of step (a), such that the new primer extension products are synthesized using both the original target sequences and the primer extension products produced in step (a) as templates, thus resulting in the amplification of the target nucleic acid sequence. PA1 a) Treating the sample with oligonucleotide complement pairs A,A' and B,B' under hybridizing and gap filling conditions. A is an oligonucleotide and A' is an oligonucleotide that is complementary to A. B is an oligonucleotide and B' is an oligonucleotide that is complementary to B. When the set of two pairs A,A' and B,B' are hybridized to nucleic acid target, A and B, which hybridize to one strand of the target forms a gap of one or more bases between them. Also, A' and B' form a gap of one or more bases between them. The gap is filled with labeled or unlabeled base(s) such that A and B become one strand with a continuous base sequence with one or more extra labeled or unlabeled bases, (A-Q-B), where Q is(are) the base(s) that fill the gap. Q could also be a base modified to be resistant to degradation caused by the 3'.fwdarw.5' exonuclease activity of polymerases used to fill the gap. Also, A' and B' are now one strand with a continuous base sequence with one or more extra labeled or unlabeled bases, (A'-Q'-B'), where Q' is(are) the base(s) that fill the gap. Similarly, Q' could also be one or more modified bases resistant to degradation caused by the 3', 5' exonuclease activity of polymerases. For any continuous base sequence A-Q-B or A'-Q'-B', Q or Q' can be composed of only one set of base pairs for any specific sequence, i.e., A-T, AU, G-C, or derivatives of these bases. A-Q-B and A'-Q'-B' are now joined, oligonucleotide products and can now serve as "target" sequences for other oligonucleotide complement pairs. When the joined, oligonucleotide product is formed by this gap-filling, ligated process it is termed an "oligonucleotide repair product." PA1 b) Treating the sample under denaturing conditions to separate the oligonucleotide repair products from their targets, if the nucleic acid target sequence(s) is(are) present. PA1 c) Treating the sample as in step (a) with oligonucleotide complement pairs A,A' and B,B' under hybridizing and gap-filling conditions such that an oligonucleotide repair product is obtained using each of the single strands produced in step (b), resulting in the amplification of the specific nucleic acid target sequence(s) if present.
Steps (a)-(c) may be conducted sequentially or simultaneously. In addition, steps (b) and (c) may be repeated until the desired level of sequence amplification is obtained. As discussed in U.S. Pat. Nos. 4,683,195 and 4,683,202, the product of step (c) may be detected using probes.
The PCR method has a disadvantage in that it fails to completely overcome the sensitivity problem. The PCR method uses all four nucleotide bases to extend the primer fragments. Therefore, extension products may be created from other, non-target nucleic acid templates that may be present in the sample such as nicked, double-stranded DNA. The use of the PCR method results in considerable background of amplified DNA other than the target sequence(s).
As will be discussed in detail later, the present invention uses at least two oligonucleotides for each strand of target nucleic acid sequence and uses fewer than all four bases, thus reducing the problem of nonspecific, background amplification for a number of reasons. For example, when labeled nucleotides are used, the gap will be filled with labeled nucleotides if the nucleic acid target sequence exists in the sample and the irrelevant sequences will not be copied or labeled.
The polymerase chain reaction method also requires heat stable enzymes for the process to be automated, while the process of the present invention can be performed using heat-labile enzymes or without any enzymes, depending upon the particular embodiment. In addition, the detection of amplified nucleic acids produced in the PCR method often requires the use of gels or a capturing system, which are laborious detection methods. In contrast, the detection of the amplified sequences in the present invention is relatively simple. For example, a Sephadex column can be used to separate the joined, oligonucleotide products formed when the target sequence is present apart from the individual nucleotides.
Other methods, beside the PCR method, exist for producing nucleic acids in large amounts from initially small amounts. For example, there is the method of subcloning a nucleic acid in the appropriate host system, where the desired nucleic acid is inserted into an appropriate vector which is used to transform the host. When the host is cultured, the vector is replicated, and hence more copies of the desired nucleic acid are produced. For a brief description of subcloning nucleic acid fragments, see Maniatis, T., et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 390-401 (1982). See also the techniques described in U.S. Pat. Nos. 4,416,988 and 4,403,036.
Other methods for synthesizing nucleic acids include the organic synthesis of a nucleic acid from nucleotide derivatives such as the methods described in U.S. Pat. No. 4,356,270. Another example of the method for synthesizing nucleic acid is provided in U.S. Pat. No. 4,293,652, which is a hybrid of organic synthesis and molecular cloning. The discussion of these and other methods in the '195 and '202 patents, as well as the patents listed above, are incorporated herein by reference.